Short Communication
Florivory and pollinator visitation: a cautionary tale Kaoru Tsuji1,2*, Manpreet K. Dhami1, David J.R. Cross1, Carolyn P. Rice1, Nic H. Romano1 and Tadashi Fukami1 1 2
Department of Biology, Stanford University, Stanford, CA 94305, USA Center for Ecological Research, Kyoto University, 2-Hirano, Otsu, Shiga 520-2113, Japan
Received: 26 February 2016; Accepted: 1 May 2016; Published: 13 May 2016 Associate Editor: Karina Boege Citation: Tsuji K, Dhami MK, Cross DJR, Rice CP, Romano NH, Fukami T. 2016. Florivory and pollinator visitation: a cautionary tale. AoB PLANTS 8: plw036; doi:10.1093/aobpla/plw036
Abstract. Florivory, or damage to flowers by herbivores, can make flowers less attractive to pollinators, potentially resulting in reduced plant fitness. However, not many studies have combined observations with experiments to assess the causal link between florivory and pollination. We conducted field observations at eight sites in northern California, combined with field experiments that involved artificial floral damage, to study the effect of florivory on pollination in the hummingbird-pollinated sticky monkeyflower, Mimulus aurantiacus. We used two indicators of pollinator visitation, stigma closure and the presence of microorganisms in floral nectar. The field observations revealed that stigma closure was less frequent in damaged flowers than in intact flowers. In the experiments, however, floral damage did not decrease stigma closure or microbial detection in nectar. Instead, neighbouring flowers were similar for both indicators. These results suggest that the observed negative association between florivory and pollination is not causal and that the location of flowers is more important to pollinator visitation than florivory in these populations of M. aurantiacus.
Keywords:
Flower; hummingbirds; Mimulus aurantiacus; nectar microbes; petal herbivory; stigma closure.
Introduction Floral herbivory can be as widespread as foliar herbivory, but its potential effects on plant fitness have only recently begun to be investigated (McCall and Irwin 2006; Maldonado et al. 2015). Florivory often reduces flower size (Strauss and Whittall 2006) and nectar production (Krupnick et al. 1999; Strauss and Whittall 2006), both of which may reduce pollinator visitation as many pollinators tend to prefer larger flowers and greater nectar production (Bell 1985; Galen 1989; Kudoh and Whigham 1998; Krupnick et al. 1999; Arista and Ortiz 2007; Shumitt 2014). An increasing number of studies suggest that
floral damage can indeed decrease pollinator visitation (Karban and Strauss 1993; Pohl et al. 2006; Ashman and Penet 2007; Penet et al. 2009; Cardel and Koptur 2010; ~ ber et al. 2010; Cares-Sua rez et al. 2011), potentially So resulting in reduced pollination and plant fitness (Krupnick and Weis 1999; Mothershead and Marquis 2000; Leavitt and Robertson 2006; McCall and Irwin nchez-Lafuente 2006; Strauss and Whittall 2006; Sa 2007; Carezza et al. 2011). However, studies on florivory have often used either field observations or experiments, not both (but see examples of using both in McCall 2008, 2010). Combined use of
* Corresponding author’s e-mail address: [email protected] Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
AoB PLANTS www.aobplants.oxfordjournals.org
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Tsuji et al. — Florivory and pollinator visitation
observations and experiments is needed in order to determine (i) if a relationship exists between florivory and pollination in natural populations, through observations, and (ii) if the observed florivory–pollination relationship is causal, through experiments. In this paper, we report a study that examined whether florivory was related to pollinator visitation through a combination of field observations and experiments. We first observed floral damage (primarily to petals) and stigma closure, an indicator of pollination in the sticky monkeyflower, Mimulus aurantiacus, at eight sites across an 200 km geographic region, to investigate the relationship between florivory and pollinator visitation. The data revealed a negative association. To examine if this association was causal, we then conducted field experiments, in which M. aurantiacus flowers were artificially damaged and two indicators of pollinator visitation recorded, stigma closure and the presence of microorganisms in nectar.
Methods Species description Mimulus aurantiacus is a hummingbird-pollinated perennial and self-compatible shrub native to California and Oregon (Fetscher and Kohn 1999; Vannette et al. 2013). The stigma of M. aurantiacus closes upon contact and stays closed if much pollen is received, but reopens if no or little pollen is received (Fetscher and Kohn 1999). For this reason, stigma closure can be used as an indicator of pollinator visitation in this species (Peay et al. 2012; Vannette et al. 2013). Furthermore, many of the microorganisms that are found in M. aurantiacus nectar are introduced to flowers primarily via hummingbirds (Belisle et al. 2012). Thus, the presence of microorganisms in floral nectar can also be used as an indicator of pollinator visitation. To estimate the age of the flowers, we used the condition of stamens as a proxy. Mimulus flowers have four stamens per flower, which dehisce and deteriorate as the flowers age. Young flowers (most likely 1–2 days old) have undehisced yellow stamens, while middle-aged flowers (typically 3–5 days old) have orange-brown dehisced stamens. Older flowers (typically 6–8 days old) have dark brown and shrunken or degenerate stamens. Stamen condition based on this distinction was used to categorize flowers into estimated age groups.
Field observations We recorded stigma closure (open or closed), flower damage (observed primarily on petals, and not the rest of the floral organs, including the stigma) and the age of flowers (young, middle-aged or old) from a total of 500 haphazardly selected flowers on 60 individuals at 8 sites
002
AoB PLANTS www.aobplants.oxfordjournals.org
in northern California (Fig. 1 and Table 1), between 30 June and 16 July 2015. We recorded the extent of floral damage for each flower we observed (i.e. intact, partially damaged, half damaged, heavily damaged), but we did not find any significant effect of the floral damage extent on pollination, so we focused on the presence or the absence of damage (i.e. intact or damaged) in the analyses presented in this paper. For data collection at each site, we haphazardly chose plants along roads and selected 6–15 flowers from each of the plants. We did not directly confirm that all of the damage on each flower we sampled for this study was actually caused by florivores. However, our extensive observations at one site (Jasper Ridge Biological Preserve of Stanford University) indicated that many, if not all, instances of floral damage, which left holes and bits of variable sizes (Fig. 2A), were caused by insect florivores, such as katydids, grasshoppers and lepidopteran larvae. The data were analysed using R (3.11 version, The R foundation for Statistical Computing Platform). We used a generalized linear mixed model (GLMM) with binomial distribution and a logistic function in lme4 package, and used the likelihood ratio test in order to determine whether stigma closure was significantly associated with flower damage and age. In the model, we used stigma closure as the response variable, flower age, flower damage, and the interaction of the age and the damage as fixed predictors, and shrub individuals and sites as random effects. In addition, we also used a similar GLMM to determine whether flower damage significantly differed among individuals and among sites. In this model, we used flower damage as the response variable, flower age as fixed predictors, and shrub individual and site as random effects. Finally, we also used a regression analysis to test whether the overall proportion of flowers that had a closed stigma at a site was significantly correlated with the frequency of flowers that had natural damage at the matching site. For this regression, we focused on old flowers.
Field experiments We marked a total of 236 pairs of flowers that were located close to each other (within 30 cm) on a total of 83 plants and artificially damaged on one of each of the paired flowers. Artificial florivory was intended to mimic a severe level of naturally observed florivory on petals (Fig. 2B). This experiment was conducted at site SB (Fig. 1) and at a common garden at the Stock Farm plant growth facility on the Stanford University campus in Stanford, CA, USA. At the SB site, we used young flowers for this experiment, conducted from 24 to 28 July 2015. At the common garden, the experiment was repeated nine times from 4 to 28 August 2015, using both young
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Tsuji et al. — Florivory and pollinator visitation
Sacramento BB
Sacramento BB
MW San Francisco
MW
SR San Francisco
SB
SR
San Jose
SB
SA
San Jose SA
JP
Pacific Ocean
CH BS
JP CH
150 km 100 mi
© d-maps.com
BS
San Luis Obispo 150 km
San Luis Obispo
Figure 1. Site of observations (see Table 1 for coordinates).
and middle-aged flowers. At both sites, we observed hummingbirds (Calypte anna) frequently visiting M. aurantiacus flowers. For each pair of flowers, 4 days after making artificial damage, we recorded stigma closure and additional natural damage on the flowers, and then collected the flowers. From each of the collected flowers, we extracted nectar using a 10-mL microcapillary tube and delivered the nectar into 40 mL of sterile water. The diluted nectar samples were further diluted and plated on yeast malt agar (YMA; Difco, Sparks, MD, USA) supplemented with 100 mg mL1 of the antibacterial chloramphenicol. After 5 days of incubation at 25 C, we checked for the presence or the absence of microbial colonies on the agar plates. Chloramphenicol was used so as to focus on the presence of yeast, such as Metschnikowia reukaufii, rather than bacteria, in nectar. In M. aurantiacus, we have previously found that yeasts appeared more dependent on hummingbirds for nectar colonization than bacteria (Belisle et al. 2012). The presence of yeasts in nectar therefore likely serves as a better indicator of pollinator
AoB PLANTS www.aobplants.oxfordjournals.org
visitation than that of bacteria. However, our previous work with molecular identification of colonies has also indicated that some bacterial taxa may be capable of forming colonies on chloramphenicol-supplemented YMA and that these bacterial colonies tend to be distinctly smaller than yeast colonies. For this reason, we also analysed the presence of large colonies, but the results were almost identical regardless of whether we considered all colonies or only large colonies. In this paper, we report results for all colonies. Data on the frequency of flowers from which microbial colonies were detected were analysed by v2 test and Fisher’s exact test in R, in order to determine if artificial damage affected the frequency of stigma closure or the occurrence of microorganisms in nectar. In addition, we also used v2 test and Fisher’s exact test to determine whether the paired flowers were more similar to each other at the end of the experiment than expected by chance in their pollination status. Furthermore, we used GLMM with binomial distribution and a logistic function in lme4 package, and used the likelihood ratio test in order
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Tsuji et al. — Florivory and pollinator visitation
Table 1. Observed number of plants and flowers at eight sites. Site
Coordinates
No. of observed
No. of observed
plants
flowers
A
...................................................................................................... BB
38.20, 123.02, 25
13
MW
37.53, 122.34, 184
10
98
SR
37.37, 122.27, 216
6
47
SB
37.29, 122.21, 335
12
93
SA
37.05, 122.15, 136
4
32
JP
36.34, 121.51, 196
4
31
CH
36.24, 121.54, 51
6
47
BS
36.20, 121.53, 106
5
39
60
500
Total
113
to determine whether stigma closure or microbial detection was significantly associated with flower damage. In this model, we used stigma closure or microbial detection as the response variable, flower age, flower damage and the interaction of age and damage as fixed predictors, and pair ID and site as random effects. For these analyses, we excluded pairs in which any natural damage was observed on the initially intact flower at the time of data collection. The results were qualitatively the same, however, when we included these pairs in the analyses.
Results Field observations Stigma closure was significantly related to flower age, flower damage, their interaction, shrub individual and observation site (Table 2). Frequency of stigma closure increased significantly with increasing flower age and was significantly lower in damaged flowers (34.5 %) than in intact flowers (76.4 %) when old flowers were observed (Fig. 3). Similarly, the likelihood of observing flower damage itself varied significantly with flower age, shrub individual and observation site [see Supporting Information – Table 1 and Fig. 1]). At the site scale, the frequency of stigma closure was negatively correlated with the proportion of floral damage (t ¼ 3.39, P ¼ 0. 019 [see Supporting Information – Fig. 2]).
Field experiments Floral damage did not significantly decrease stigma closure or the detection of microbes in nectar (stigma: odds ratio ¼ 0.36, df ¼ 1, P ¼ 0.55; microbes: odds ratio ¼ 0.19, df ¼ 1, P ¼ 0.66, Tables 3 and 4). This pattern was observed at both sites (stigma: odds ratio ¼ 0.42, 0, df ¼ 1, 1, P ¼ 0.52, 1; microbes: odds ratio ¼ 0, 0.56, df ¼ 1, 1,
004
AoB PLANTS www.aobplants.oxfordjournals.org
Natural damage
B
Intact flower
Artificial damage Figure 2. Photographs showing (A) a flower with natural damage by florivorous insects and (B) an example of paired flowers, with one intact and one experimentally damaged. Photo credit: P. Garvey and M. Dhami.
P ¼ 1, 0.45, at the SB site and the Stock Farm site, respectively, [see Supporting Information – Tables 2–5]. However, paired flowers were similar in both stigma closure and the presence of microbes. That is, if a flower had a closed stigma, it was significantly more likely that the paired flower also had a closed, than open, stigma (odds ratio ¼ 9.05, df ¼ 1, P ¼ 0.0026, Table 5). Likewise, if microbes were detected from a flower, it was significantly likely that they were also detected from the paired flower (odds ratio ¼ 49.26, df ¼ 1, P < 0.0001, Table 6). Consistent with these results, the GLMM also indicated that stigma closure and microbial detection were significantly related to pair ID, but not to flower damage [see Supporting Information – Tables 10 and 11. When analysed separately for the two sites, the pattern was significant for stigma closure at SB (odds ratio ¼ 12.3, df ¼ 1, P ¼ 0.0005 [see Supporting Information – Table 6]), but not at Stock Farm (odds ratio ¼ 0, df ¼ 1, P ¼ 1 [see Supporting Information – Table 7]), and it was significant for microbial detection at Stock Farm (odds
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Tsuji et al. — Florivory and pollinator visitation
Table 2. Analytical results of field observations using the likelihood ratio test.
Table 3. Stigma closure of intact and experimentally damaged flowers.
Predictor
......................................................................................................
df
Likelihood
P value
......................................................................................................
Flower age
2
68.13
Florivory and pollinator visitation: a cautionary tale Kaoru Tsuji1,2*, Manpreet K. Dhami1, David J.R. Cross1, Carolyn P. Rice1, Nic H. Romano1 and Tadashi Fukami1 1 2
Department of Biology, Stanford University, Stanford, CA 94305, USA Center for Ecological Research, Kyoto University, 2-Hirano, Otsu, Shiga 520-2113, Japan
Received: 26 February 2016; Accepted: 1 May 2016; Published: 13 May 2016 Associate Editor: Karina Boege Citation: Tsuji K, Dhami MK, Cross DJR, Rice CP, Romano NH, Fukami T. 2016. Florivory and pollinator visitation: a cautionary tale. AoB PLANTS 8: plw036; doi:10.1093/aobpla/plw036
Abstract. Florivory, or damage to flowers by herbivores, can make flowers less attractive to pollinators, potentially resulting in reduced plant fitness. However, not many studies have combined observations with experiments to assess the causal link between florivory and pollination. We conducted field observations at eight sites in northern California, combined with field experiments that involved artificial floral damage, to study the effect of florivory on pollination in the hummingbird-pollinated sticky monkeyflower, Mimulus aurantiacus. We used two indicators of pollinator visitation, stigma closure and the presence of microorganisms in floral nectar. The field observations revealed that stigma closure was less frequent in damaged flowers than in intact flowers. In the experiments, however, floral damage did not decrease stigma closure or microbial detection in nectar. Instead, neighbouring flowers were similar for both indicators. These results suggest that the observed negative association between florivory and pollination is not causal and that the location of flowers is more important to pollinator visitation than florivory in these populations of M. aurantiacus.
Keywords:
Flower; hummingbirds; Mimulus aurantiacus; nectar microbes; petal herbivory; stigma closure.
Introduction Floral herbivory can be as widespread as foliar herbivory, but its potential effects on plant fitness have only recently begun to be investigated (McCall and Irwin 2006; Maldonado et al. 2015). Florivory often reduces flower size (Strauss and Whittall 2006) and nectar production (Krupnick et al. 1999; Strauss and Whittall 2006), both of which may reduce pollinator visitation as many pollinators tend to prefer larger flowers and greater nectar production (Bell 1985; Galen 1989; Kudoh and Whigham 1998; Krupnick et al. 1999; Arista and Ortiz 2007; Shumitt 2014). An increasing number of studies suggest that
floral damage can indeed decrease pollinator visitation (Karban and Strauss 1993; Pohl et al. 2006; Ashman and Penet 2007; Penet et al. 2009; Cardel and Koptur 2010; ~ ber et al. 2010; Cares-Sua rez et al. 2011), potentially So resulting in reduced pollination and plant fitness (Krupnick and Weis 1999; Mothershead and Marquis 2000; Leavitt and Robertson 2006; McCall and Irwin nchez-Lafuente 2006; Strauss and Whittall 2006; Sa 2007; Carezza et al. 2011). However, studies on florivory have often used either field observations or experiments, not both (but see examples of using both in McCall 2008, 2010). Combined use of
* Corresponding author’s e-mail address: [email protected] Published by Oxford University Press on behalf of the Annals of Botany Company. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
AoB PLANTS www.aobplants.oxfordjournals.org
C The Authors 2016 V
100
Tsuji et al. — Florivory and pollinator visitation
observations and experiments is needed in order to determine (i) if a relationship exists between florivory and pollination in natural populations, through observations, and (ii) if the observed florivory–pollination relationship is causal, through experiments. In this paper, we report a study that examined whether florivory was related to pollinator visitation through a combination of field observations and experiments. We first observed floral damage (primarily to petals) and stigma closure, an indicator of pollination in the sticky monkeyflower, Mimulus aurantiacus, at eight sites across an 200 km geographic region, to investigate the relationship between florivory and pollinator visitation. The data revealed a negative association. To examine if this association was causal, we then conducted field experiments, in which M. aurantiacus flowers were artificially damaged and two indicators of pollinator visitation recorded, stigma closure and the presence of microorganisms in nectar.
Methods Species description Mimulus aurantiacus is a hummingbird-pollinated perennial and self-compatible shrub native to California and Oregon (Fetscher and Kohn 1999; Vannette et al. 2013). The stigma of M. aurantiacus closes upon contact and stays closed if much pollen is received, but reopens if no or little pollen is received (Fetscher and Kohn 1999). For this reason, stigma closure can be used as an indicator of pollinator visitation in this species (Peay et al. 2012; Vannette et al. 2013). Furthermore, many of the microorganisms that are found in M. aurantiacus nectar are introduced to flowers primarily via hummingbirds (Belisle et al. 2012). Thus, the presence of microorganisms in floral nectar can also be used as an indicator of pollinator visitation. To estimate the age of the flowers, we used the condition of stamens as a proxy. Mimulus flowers have four stamens per flower, which dehisce and deteriorate as the flowers age. Young flowers (most likely 1–2 days old) have undehisced yellow stamens, while middle-aged flowers (typically 3–5 days old) have orange-brown dehisced stamens. Older flowers (typically 6–8 days old) have dark brown and shrunken or degenerate stamens. Stamen condition based on this distinction was used to categorize flowers into estimated age groups.
Field observations We recorded stigma closure (open or closed), flower damage (observed primarily on petals, and not the rest of the floral organs, including the stigma) and the age of flowers (young, middle-aged or old) from a total of 500 haphazardly selected flowers on 60 individuals at 8 sites
002
AoB PLANTS www.aobplants.oxfordjournals.org
in northern California (Fig. 1 and Table 1), between 30 June and 16 July 2015. We recorded the extent of floral damage for each flower we observed (i.e. intact, partially damaged, half damaged, heavily damaged), but we did not find any significant effect of the floral damage extent on pollination, so we focused on the presence or the absence of damage (i.e. intact or damaged) in the analyses presented in this paper. For data collection at each site, we haphazardly chose plants along roads and selected 6–15 flowers from each of the plants. We did not directly confirm that all of the damage on each flower we sampled for this study was actually caused by florivores. However, our extensive observations at one site (Jasper Ridge Biological Preserve of Stanford University) indicated that many, if not all, instances of floral damage, which left holes and bits of variable sizes (Fig. 2A), were caused by insect florivores, such as katydids, grasshoppers and lepidopteran larvae. The data were analysed using R (3.11 version, The R foundation for Statistical Computing Platform). We used a generalized linear mixed model (GLMM) with binomial distribution and a logistic function in lme4 package, and used the likelihood ratio test in order to determine whether stigma closure was significantly associated with flower damage and age. In the model, we used stigma closure as the response variable, flower age, flower damage, and the interaction of the age and the damage as fixed predictors, and shrub individuals and sites as random effects. In addition, we also used a similar GLMM to determine whether flower damage significantly differed among individuals and among sites. In this model, we used flower damage as the response variable, flower age as fixed predictors, and shrub individual and site as random effects. Finally, we also used a regression analysis to test whether the overall proportion of flowers that had a closed stigma at a site was significantly correlated with the frequency of flowers that had natural damage at the matching site. For this regression, we focused on old flowers.
Field experiments We marked a total of 236 pairs of flowers that were located close to each other (within 30 cm) on a total of 83 plants and artificially damaged on one of each of the paired flowers. Artificial florivory was intended to mimic a severe level of naturally observed florivory on petals (Fig. 2B). This experiment was conducted at site SB (Fig. 1) and at a common garden at the Stock Farm plant growth facility on the Stanford University campus in Stanford, CA, USA. At the SB site, we used young flowers for this experiment, conducted from 24 to 28 July 2015. At the common garden, the experiment was repeated nine times from 4 to 28 August 2015, using both young
C The Authors 2016 V
Tsuji et al. — Florivory and pollinator visitation
Sacramento BB
Sacramento BB
MW San Francisco
MW
SR San Francisco
SB
SR
San Jose
SB
SA
San Jose SA
JP
Pacific Ocean
CH BS
JP CH
150 km 100 mi
© d-maps.com
BS
San Luis Obispo 150 km
San Luis Obispo
Figure 1. Site of observations (see Table 1 for coordinates).
and middle-aged flowers. At both sites, we observed hummingbirds (Calypte anna) frequently visiting M. aurantiacus flowers. For each pair of flowers, 4 days after making artificial damage, we recorded stigma closure and additional natural damage on the flowers, and then collected the flowers. From each of the collected flowers, we extracted nectar using a 10-mL microcapillary tube and delivered the nectar into 40 mL of sterile water. The diluted nectar samples were further diluted and plated on yeast malt agar (YMA; Difco, Sparks, MD, USA) supplemented with 100 mg mL1 of the antibacterial chloramphenicol. After 5 days of incubation at 25 C, we checked for the presence or the absence of microbial colonies on the agar plates. Chloramphenicol was used so as to focus on the presence of yeast, such as Metschnikowia reukaufii, rather than bacteria, in nectar. In M. aurantiacus, we have previously found that yeasts appeared more dependent on hummingbirds for nectar colonization than bacteria (Belisle et al. 2012). The presence of yeasts in nectar therefore likely serves as a better indicator of pollinator
AoB PLANTS www.aobplants.oxfordjournals.org
visitation than that of bacteria. However, our previous work with molecular identification of colonies has also indicated that some bacterial taxa may be capable of forming colonies on chloramphenicol-supplemented YMA and that these bacterial colonies tend to be distinctly smaller than yeast colonies. For this reason, we also analysed the presence of large colonies, but the results were almost identical regardless of whether we considered all colonies or only large colonies. In this paper, we report results for all colonies. Data on the frequency of flowers from which microbial colonies were detected were analysed by v2 test and Fisher’s exact test in R, in order to determine if artificial damage affected the frequency of stigma closure or the occurrence of microorganisms in nectar. In addition, we also used v2 test and Fisher’s exact test to determine whether the paired flowers were more similar to each other at the end of the experiment than expected by chance in their pollination status. Furthermore, we used GLMM with binomial distribution and a logistic function in lme4 package, and used the likelihood ratio test in order
C The Authors 2016 V
300
Tsuji et al. — Florivory and pollinator visitation
Table 1. Observed number of plants and flowers at eight sites. Site
Coordinates
No. of observed
No. of observed
plants
flowers
A
...................................................................................................... BB
38.20, 123.02, 25
13
MW
37.53, 122.34, 184
10
98
SR
37.37, 122.27, 216
6
47
SB
37.29, 122.21, 335
12
93
SA
37.05, 122.15, 136
4
32
JP
36.34, 121.51, 196
4
31
CH
36.24, 121.54, 51
6
47
BS
36.20, 121.53, 106
5
39
60
500
Total
113
to determine whether stigma closure or microbial detection was significantly associated with flower damage. In this model, we used stigma closure or microbial detection as the response variable, flower age, flower damage and the interaction of age and damage as fixed predictors, and pair ID and site as random effects. For these analyses, we excluded pairs in which any natural damage was observed on the initially intact flower at the time of data collection. The results were qualitatively the same, however, when we included these pairs in the analyses.
Results Field observations Stigma closure was significantly related to flower age, flower damage, their interaction, shrub individual and observation site (Table 2). Frequency of stigma closure increased significantly with increasing flower age and was significantly lower in damaged flowers (34.5 %) than in intact flowers (76.4 %) when old flowers were observed (Fig. 3). Similarly, the likelihood of observing flower damage itself varied significantly with flower age, shrub individual and observation site [see Supporting Information – Table 1 and Fig. 1]). At the site scale, the frequency of stigma closure was negatively correlated with the proportion of floral damage (t ¼ 3.39, P ¼ 0. 019 [see Supporting Information – Fig. 2]).
Field experiments Floral damage did not significantly decrease stigma closure or the detection of microbes in nectar (stigma: odds ratio ¼ 0.36, df ¼ 1, P ¼ 0.55; microbes: odds ratio ¼ 0.19, df ¼ 1, P ¼ 0.66, Tables 3 and 4). This pattern was observed at both sites (stigma: odds ratio ¼ 0.42, 0, df ¼ 1, 1, P ¼ 0.52, 1; microbes: odds ratio ¼ 0, 0.56, df ¼ 1, 1,
004
AoB PLANTS www.aobplants.oxfordjournals.org
Natural damage
B
Intact flower
Artificial damage Figure 2. Photographs showing (A) a flower with natural damage by florivorous insects and (B) an example of paired flowers, with one intact and one experimentally damaged. Photo credit: P. Garvey and M. Dhami.
P ¼ 1, 0.45, at the SB site and the Stock Farm site, respectively, [see Supporting Information – Tables 2–5]. However, paired flowers were similar in both stigma closure and the presence of microbes. That is, if a flower had a closed stigma, it was significantly more likely that the paired flower also had a closed, than open, stigma (odds ratio ¼ 9.05, df ¼ 1, P ¼ 0.0026, Table 5). Likewise, if microbes were detected from a flower, it was significantly likely that they were also detected from the paired flower (odds ratio ¼ 49.26, df ¼ 1, P < 0.0001, Table 6). Consistent with these results, the GLMM also indicated that stigma closure and microbial detection were significantly related to pair ID, but not to flower damage [see Supporting Information – Tables 10 and 11. When analysed separately for the two sites, the pattern was significant for stigma closure at SB (odds ratio ¼ 12.3, df ¼ 1, P ¼ 0.0005 [see Supporting Information – Table 6]), but not at Stock Farm (odds ratio ¼ 0, df ¼ 1, P ¼ 1 [see Supporting Information – Table 7]), and it was significant for microbial detection at Stock Farm (odds
C The Authors 2016 V
Tsuji et al. — Florivory and pollinator visitation
Table 2. Analytical results of field observations using the likelihood ratio test.
Table 3. Stigma closure of intact and experimentally damaged flowers.
Predictor
......................................................................................................
df
Likelihood
P value
......................................................................................................
Flower age
2
68.13
